Resin Embedding Research Articles

Ex vivo delivery of Mirococept: A dose-finding study in pig kidney after showing a low dose is insufficient to reduce delayed graft function in human kidney.

The complement system plays a pivotal role in the pathogenesis of ischemia-reperfusion injury in solid organ transplantation. Mirococept is a potent membrane-localizing complement inhibitor that can be administered ex vivo to the donor kidney prior to transplantation. To evaluate the efficacy of Mirococept in reducing delayed graft function (DGF) in deceased donor renal transplantation, we undertook the efficacy of mirococept (APT070) for preventing ischaemia-reperfusion injury in the kidney allograft (EMPIRIKAL) trial (ISRCTN49958194). A dose range of 5-25mg would be tested, starting with 10mg in cohort 1. No significant difference between Mirococept at 10mg and control was detected; hence the study was stopped to enable a further dose saturation study in a porcine kidney model. The optimal dose of Mirococept in pig kidney was 80mg. This dose did not induce any additional histological damage compared to controls or after a subsequent 3hours of normothermic machine perfusion. The amount of unbound Mirococept postperfusion was found to be within the systemic dose range considered safe in the Phase I trial. The ex vivo administration of Mirococept is a safe and feasible approach to treat DGF in deceased donor kidney transplantation. The porcine kidney study identified an optimal dose of 80mg (equivalent to 120mg in human kidney) that provides a basis for further clinical development.

Morphology of the avian yolk sac.

The avian yolk sac is a multifunctional extraembryonic organ that serves not only as a site of nutrient (yolk) absorption, but also for early hemopoiesis, and formation of blood vessels. Although the yolk sac membrane being specialized to function as an extraembryonic absorptive organ, it is neither morphologically nor functionally part of the embryonic gut. Yolk absorption is by the phagocytic activity of the extraembryonic endoderm. I used cryohistology and resin embedding histology of complete developmental series of Japanese quail to document the development of the avian yolk sac and changes of the microscopic anatomy throughout development. This material is complemented by complete series of MRT-scans of live ostrich embryos from beginning of incubation through hatching. Considerable changes of size and shape of the yolk mass are documented and discussed as resulting from water flux from albumen to yolk associated with the biochemical activation of yolk sac proteins. During embryogenesis, the yolk sac endoderm forms villi that increase the absorptive surface and reach into the yolk ball. The histology of the absorptive epithelium is specialized for phagocytic absorption of yolk. During early developmental stages, the extraembryonic endoderm is single layered, but it eventually becomes several layers thick during later stages. The extraembryonic mesoderm forms an extensive layer of hematopoietic tissue; deep in this tissue lie the yolk sac vessels. During late stages of development, the erythropoietic tissue disappears, blood vessels are obliterated, and the yolk sac epithelium becomes apoptotic. Results are discussed in the light of the evolutionary history and phylogeny of the amniote egg.

Ultramicrotomy Analysis of Peptide-Treated Cells.

Electron microscopy offers necessary precision for the characterization of peptide materials at the nanoscale. Analysis is typically performed for acellular material specimens, whereas measurements in more complex, cellular environments prompt additional considerations for sample processing. Herein, we describe a protocol for the ultramicrotomy analysis of peptide-treated bacterial and mammalian cells. An emphasis is made on cell analysis following peptide treatment, as opposed to peptide analysisin cells, and focuses on sample processing, including fixation and staining procedures, resin embedding, sectioning, and imaging. The application of the protocol is demonstrated for intracellular measurements using antimicrobial peptide materials.

Strategies for Optimizing Heavy Metal Staining in Freeze-substituted Resin Embedded Plants Samples for Electron and X-ray Microscopy

Journal Article Strategies for Optimizing Heavy Metal Staining in Freeze-substituted Resin Embedded Plants Samples for Electron and X-ray Microscopy Get access Kirk Czymmek, Kirk Czymmek Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Keith Duncan, Keith Duncan Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Ningning Zhang, Ningning Zhang Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Ru Zhang Ru Zhang Donald Danforth Plant Science Center, Saint Louis, Missouri, United States Search for other works by this author on: Oxford Academic Google Scholar Microscopy and Microanalysis, Volume 26, Issue S2, 1 August 2020, Pages 142–143, https://doi.org/10.1017/S1431927620013550 Published: 01 August 2020

Uranium-free X solution: a new generation contrast agent for biological samples ultrastructure

Biological samples are mainly composed of elements with a low atomic number which show a relatively low electron scattering power. For Transmission Electron Microscopy analysis, biological samples are generally embedded in resins, which allow thin sectioning of the specimen. Embedding resins are also composed by light atoms, thus the contrast difference between the biological sample and the surrounding resin is minimal. Due to that reason in the last decades, several staining solutions and approaches, performed with heavy metal salts, have been developed with the purpose of enhancing both the intrinsic sample contrast and the differences between the sample and resin. The best staining was achieved with the uranyl acetate (UA) solution, which has been the election method for the study of morphology in biological samples. More recently several alternatives for UA have been proposed to get rid of its radiogenic issues, but to date none of these solutions has achieved efficiencies comparable to UA. In this work, we propose a different staining solution (X Solution or X SOL), characterized by lanthanide polyoxometalates (LnPOMs) as heavy atoms source, which could be used alternatively to UA in negative staining (NS), in en bloc staining, and post sectioning staining (PSS) of biological samples. Furthermore, we show an extensive chemical characterization of the LnPOM species present in the solution and the detailed work for its final formulation, which brought remarkable results, and even better performances than UA.

Geographic Atrophy: Confocal Scanning Laser Ophthalmoscopy, Histology, and Inflammation in the Region of Expanding Lesions.

PurposeTo describe the pathology of AMD in eyes with geographic atrophy (GA) using confocal scanning laser ophthalmoscopy (SLO) blue light autofluorescence (BAF), and near-infrared (IR) AF and to correlate it with the histology and immunohistochemistry analysis at the margins of the GA lesion.MethodsEnucleated, fixed eyes from seventeen donors with GA were imaged and analyzed by BAF-SLO, IRAF-SLO, and by fundus macroscopy (FM). Tissue from the margins of the GA lesions was cut and processed for resin embedding and histology or cryosectioning and fluorescence in the green and far-red channels, and immunohistochemistry to assess markers of inflammation. Isolated DNA from donors was genotyped for single nucleotide polymorphisms (SNPs) previously shown to be risk factors for the development and progression of AMD.ResultsAround the leading edge of the GA lesions we observed hypertrophic RPE cells with cytoplasm filled with granules fluorescent both in the far-red and green-red channels; abundant microglia and macrophage; deposition of complement factor H (CFH) in Bruch's membrane (BM) and increased membrane attack complex (MAC) on RPE cells.ConclusionsFluorescence imaging of cryosections of RPE cells around the leading edge of the GA lesions suggest that IRAF-SLO visualizes mostly melanin-related compounds. In addition, medium-size GA atrophy displayed the most significant changes in inflammation markers.

Effect of smear layer removal agents on the microhardness and roughness of radicular dentin

PurposeTo evaluate the effect of phytic acid (IP6) on the surface roughness and microhardness of human root canal dentin and compare it to other smear layer removal agents. Materials and methodsFifty extracted human maxillary incisors were sectioned longitudinally into a total of 100 specimens followed by embedding in auto-polymerizing acrylic resin. The specimens were polished and then randomly divided into five groups (n = 20) according to the test solution used to condition root canal dentin: 17% ethylenediaminetetraacetic acid (EDTA); 10% citric acid (CA); 1% IP6; 37% phosphoric acid (PA); or distilled water (control group). Each specimen was treated with a total volume of 1 ml of each solution for 1 min with agitation. Each group was then divided into two subgroups of 10 specimens each. The specimens of the first subgroup were used to determine microhardness, using Vickers hardness tester, and the specimens of the second subgroup were used to measure surface roughness, using a confocal laser scanning microscope. The results were analyzed statistically using one-way ANOVA and Tukey tests, α = 0.05. ResultsAll the tested groups exhibited microhardness and surface roughness values that were statistically significantly different when compared with the control group (P < 0.05). The microhardness value obtained with IP6 was significantly lower when compared to EDTA, CA, and the control group, whereas its roughness value was significantly higher compared to the aforementioned groups. However, there was no significant difference between IP6 and PA (P > 0.05). ConclusionsIP6 and PA showed the lowest microhardness and the highest surface roughness values.

Slicing Spheroids in Microfluidic Devices for Morphological and Immunohistochemical Analysis.

Microfluidic devices utilizing spheroids play important roles in in vitro experimental systems to closely simulate morphological and biochemical characteristics of the in vivo tumor microenvironment. For the observation and analysis of the inner structure of spheroids, sectioning is an efficient approach. However, conventional microfluidic devices are difficult for sectioning, and therefore, spheroids inside the microfluidic channels have not been sliced well. We proposed a microfluidic device created from embedding resin for sectioning. Spheroids were cultured, embedded by resin, and sectioned in the microfluidic device. Slices of the sectioned spheroids yielded clear images at the cellular level. According to morphological and immunohistochemical analyses of the slices of the spheroid, specific protein distribution was observed.

Observation of exopolysaccharides (EPS) from Lactobacillus helveticus SBT2171 using the Tokuyasu method.

Some species of lactic acid bacteria used for the production of natural cheese produce exopolysaccharides (EPS). Electron microscopy is useful for analyzing the microstructure of EPS produced by lactic acid bacteria. However, pretreatments used to observe the microstructure of EPS by electron microscopy, such as dehydration and resin embedding, can result in EPS flowing out easily from the cell. Therefore, in this study, the Tokuyasu method was conducted on cryosection to reduce EPS outflow. Two types of observation method, namely, using lectin and ruthenium red, were conducted in an attempt to observe EPS produced by Lactobacillus helveticus SBT2171. Observation using the lectin method confirmed that colloidal gold particles conjugated with a lectin recognizing β-galactoside were present in the capsule. Structures that appeared to be β-galactoside-containing slime polysaccharides that were released from the cell wall were also observed. Observation using ruthenium red showed that capsular polysaccharides (CPS) in the capsule were present as a net-like structure. Colloidal gold conjugation with an anti-β-lactoglobulin antibody, in addition to ruthenium red staining, allowed the identification of slime polysaccharides released from the cell wall in the milk protein network derived from the culture medium. Based on these results, the Tokuyasu method was considered to be a useful pretreatment method to clarify and observe the presence of EPS. In particular, both CPS in the capsule and slime exopolysaccharides released from the cell wall were visualized.

A simple method based on confocal microscopy and thick sections recognizes seven subphases in growth plate chondrocytes

This manuscript reports a novel procedure to imaging growth plate chondrocytes by using confocal microscopy. The method is based on fixed undecalcified bone samples, in-block staining with eosin, epoxy resin embedding and grinding to obtain thick sections. It is simple, inexpensive and provides three-dimensional images of entire chondrocytes inside their native lacunae. Quantitative analysis of volume, shape and cytoplasm density of chondrocytes at different strata of the growth plate allowed to objectively grade chondrocytes of the growth plate in seven different clusters. These seven categories of chondrocytes were subsequently evaluated by immunohistochemistry of some well-defined molecular landmarks of chondrocyte differentiation. Furthermore, immunohistochemical analysis of proteins responsible for ionic changes and water transport allowing chondrocyte swelling during hypertrophy was also performed. Results obtained indicate that four subphases can be defined in the pre-hypertrophic zone and three subphases in the hypertrophic zone, a fact that raises that chondrocytes of the growth plate are less homogeneous than usually considered when different zones are defined according to subjective cell morphological criteria. Results in the present study provide a technological innovation and gives new insights into the complexity of the process of chondrocyte differentiation in the growth plate.

Multimodal Characterization of Resin Embedded and Sliced Polymer Nanoparticles by Means of Tip-Enhanced Raman Spectroscopy and Force-Distance Curve Based Atomic Force Microscopy.

Understanding the property-function relation of nanoparticles in various application fields involves determining their physicochemical properties, which is still a remaining challenge to date. While a multitude of different characterization tools can be applied, these methods by themselves can only provide an incomplete picture. Therefore, novel analytical techniques are required, which can address both chemical functionality and provide structural information at the same time with high spatial resolution. This is possible by using tip-enhanced Raman spectroscopy (TERS), but due to its limited depth information, TERS is usually restricted to investigations of the nanoparticle surface. Here, TERS experiments are established on polystyrene nanoparticles (PS NPs) after resin embedding and microtome slicing. With that, unique access to their internal morphological features is gained, and thus, enables differentiation between information obtained for core- and shell-regions. Complementary information is obtained by means of transmission electron microscopy (TEM) and from force-distance curve based atomic force microscopy (FD-AFM). This multimodal approach achieves a high degree of discrimination between the resin and the polymers used for nanoparticle formulation. The high potential of TERS combined with advanced AFM spectroscopy tools to probe the mechanical properties is applied for quality control of the resin embedding procedure.

Novel insights on the study of a fifteenth-century oro di metà/Zwischgold gilding by means of ion and electron microscopy: characterization of the stratigraphy avoiding cross-sections preparation

Abstract In this work, an analytical methodology to identify and thoroughly characterize the oro di meta/Zwischgold gilding is reported. Gildings in paintings are commonly studied by means of optical microscopy (OM), Scanning Electron Microscopy (SEM) coupled with Energy Dispersive Spectrometry (SEM-EDS), as well as other techniques, but they all require sample preparation. In this study, a sample of Zwischgold taken from an Italian panel painting of the fifteenth century was thoroughly characterized using Focused Ion Beam (FIB)-SEM sectioning, accessing the layered structure from the surface into depth without further sample preparation. The results achieved were compared to those of a further sample taken from the same area, which was prepared by conventional resin embedding and polishing techniques in order to evaluate the effects of the mechanical stresses induced by resin drying and polishing along the latter process. A more uniform morphology and sharper outlines of the metal layers were observed after FIB milling, likely due to the more accurate and less invasive cutting process. Moreover, since SEM-EDS analysis is limited in terms of resolution to be performed straight on the milled area relative to the thinness of the metal layers, Time of Flight – Secondary Ion Mass Spectrometry (ToF-SIMS) analysis was performed, and allowed revealing two distinct metal layers in the gilding consisting of Au and Ag thus confirming a Zwischgold structure.

Evaluation of secondary ions related to plant tissue using least absolute shrinkage and selection operator.

With regard to life sciences, it is important to understand biological functions such as metabolic reactions at the cellular level. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) that can provide chemical mappings at 100 nm lateral resolutions is useful for obtaining three-dimensional maps of biological molecules in cells and tissues. TOF-SIMS spectra generally contain several hundred to several thousand secondary ion peaks that provide detailed chemical information. In order to manage such a large number of peaks, data analysis methods such as multivariate analysis techniques have been applied to TOF-SIMS data of complex samples. However, the interpretation of the data analysis results is sometimes still difficult, especially for biological samples. In this study, TOF-SIMS data of resin-embedded plant samples were analyzed using one of the sparse modeling methods, least absolute shrinkage and selection operator (LASSO), to directly select secondary ions related to biological structures such as cell walls and nuclei. The same sample was measured by optical microscopy and the same measurement area as TOF-SIMS was extracted in order to prepare a target image for LASSO. The same area of the TOF-SIMS and microscope data were fused to evaluate the influence of the image fusion on the TOF-SIMS spectrum information using principal component analysis. Specifically, the authors examined onion mycorrhizal root colonized with Gigaspora margarita (an arbuscular mycorrhizal fungus). The results showed that by employing this approach using LASSO, important secondary ions from biological samples were effectively selected and could be clearly distinguished from the embedding resin.

Optical Microscopy and Electron Microscopy for the Morphological Evaluation of Tendons: A Mini Review.

The morphological characteristics of tendons have been thoroughly evaluated via microscopy. Optical microscopy and electron microscopy are the most commonly used techniques for tendon tissue observation. According to the principles of both microscopy types, preparation and evaluation methods vary. Simple optical microscopy is commonly used in the observation of cells and extracellular matrix, and many stains, including hematoxylin–eosin, Van Gieson, Prussian blue, Alcian blue, and toluidine blue, are used for evaluating cells, collagen fiber arrangement, and noncollagenous proteins. Histological scoring systems have been used in many studies for semi‐quantification. Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) are the most commonly used electron microscopy types, and special consideration is needed for the fixation and embedding protocols. Glutaraldehyde followed by osmium is most commonly used in the chemical fixation of tendon tissue, followed by epoxy resin embedment. Longitudinal sections captured in SEM images show the arrangement of collagen fibrils and the cells and lipid drops among them, while cross sections captured in TEM images show the diameter and distribution of collagen fibrils. SEM and TEM are used together for comprehensive evaluations. This mini review is focused on the preparation methodology and related evaluation indexes for the morphological evaluation of tendons.

Sample Preparation Method of Scanning and Transmission Electron Microscope for the Appendages of Woodboring Beetle.

This report described sample preparation methods that scanning and transmission electron microscope observations, demonstrated by preparing appendages of the woodboring beetle, Chlorophorus caragana Xie & Wang (2012), for both types of electron microscopy. The scanning electron microscopy (SEM) sample preparation protocol was based on sample chemical fixation, dehydration in a series of ethanol baths, drying, and sputter-coating. By adding Tween 20 (Polyoxyethylene sorbitan laurate) to the fixative and the wash solution, the insect body surface of woodboring beetle was washed more cleanly in SEM. This study's transmission electron microscopy (TEM) sample preparation involved a series of steps including fixation, ethanol dehydration, embedding in resin, positioning using fluorescence microscopy, sectioning, and staining. Fixative with Tween 20 enabled penetrate the insect body wall of woodboring beetle more easily than it would had been without Tween 20, and subsequently better fixed tissues and organs in the body, thus yielded clear transmission electron microscope observations of insect sensilla ultrastructures. The next step of this preparation was determining the positions of insect sensilla in the sample embedded in the resin block by using fluorescence microscopy to increase the precision of target sensilla positioning. This improved slicing accuracy.

Histological, Histomorphometrical, and Biomechanical Studies of Bone-Implanted Medical Devices: Hard Resin Embedding

The growing incidence of degenerative musculoskeletal disorders as well as lifestyle changes has led to an increase in the surgical procedures involving implanted medical devices in orthopedics. When studying implant/tissue interface in hard materials (i.e., metals or dense plastics) and/or in large bone segments, the hard plastic embedding of the intact undecalcified tissue envelope with the implant in situ is needed. The aim of this work is to describe the advances and the possibilities of high-temperature methyl methacrylate (MMA) embedding for the histological, histomorphometrical, and biomechanical assessment of bone-implanted medical devices. Unlike routine techniques, undecalcified bone processing histology, using high-temperature MMA, requires a complex and precise sample processing methodology and the availability of sophisticated equipment and software for both sample preparation and analyses. MMA embedding permits the evaluation of biological responses to the presence of implanted medical devices without implant removal, allowing simultaneous qualitative and quantitative histological evaluation, both static and dynamic histomorphometry, and biomechanical analyses not possible with tissue decalcification. MMA embedding, despite being a demanding procedure, is still preferred to other kinds of resin-based embedding because of its peculiar characteristics, which allow the study of samples of big dimensions also implanted with hard materials without reducing the sample or removing the material. Dynamic measurements are allowed together with biomechanical investigations at the bone-biomaterial interface, obtaining a comprehensive and precise evaluation of the safety and effectiveness of medical devices for orthopedic regenerative, reconstructive, and reparative surgery.

Influence of unmodified and modified sutures on experimental abdominal adhesive process

Experimental assessment of the effect of modified and unmodified surgical suture material on abdominal adhesive process. The study was performed on male rats of the Wistar subpopulation. There were 5 animals in each group. In all animals, midline abdominal incision was followed by suturing the parietal peritoneum with modified and unmodified suture material. All animals were euthanized with carbon dioxide vapors in 14 days after surgery. Macro- and microscopic assessment of severity of abdominal adhesive process was carried out. Two types of preparation of excised complexes 'peritoneum-suture material-adhesion' were applied for histological examination: paraffin sections and embedding in epoxy resin. Specimens were stained by Van Gieson and with methylene blue solution. Histological specimens were examined using Axio Imager A1 light microscope (Zeiss, Germany). Polypropylene filaments result extensive adhesions occupying about 75% of the area. Adhesions have a dense structure with signs of vascularization. Modification of suture material with solution of polyhydroxybutyrate/hydroxyvalerate and heparin reduce severity of adhesions. The use of modified suture material was followed by adhesions with more loose structure, no signs of vascularization. Adhesions occupied less than 25% of the area. Histological examination of excised complexes 'peritoneum-suture material-adhesion' revealed accumulation of inflammatory cells around the unmodified suture material, while there were no signs of tissue inflammatory process around the modified sutures. Application of polyhydroxybutyrate/hydroxyvalerate and heparin on the surface of surgical sutures is an effective method for prevention of abdominal adhesions.

STRUCTURE OF CALCIFICATES IN HUMAN CAROTID ARTERY ATHEROSCLEROTIC PLAQUES BY MEANS OF BACKSCATTERED SCANNING ELECTRON MICROSCOPY

Aim of the study was to investigate the atherosclerotic calcification employing our original technique of tissue staining, embedding, and backscattered scanning electron microscopy. Materials and methods . Atherosclerotic plaques excised during the carotid endarterectomy have been fixed in 10 % neutral phosphate buffered saline formalin for 24 hours, stained in 2 % osmium tetroxide for 60 hours and in alcoholic uranyl acetate for 5 hours with the subsequent epoxy resin embedding, grinding, polishing, lead citrate counterstaining for 7 minutes, sputter coating with carbon and backscattered scanning electron microscopy. We then analysed localisation, structure, and microenvironment of the calcium deposits. Results . Within the atherosclerotic plaques, we identified 3 distinct calcification morphologies: compact homogenous macrocalcifications, sheet-like heterogeneous macrocalcifications along the destructed collagen and elastin fibers, and microcalcifications around large calcium deposits with the sharp margins. However, even relatively homogenous compact macrocalcifications had uneven distribution of electron density. In conjunction with groups of mineral deposits of distinct calcium phosphate phases including nascent calcified loci, this testified to ongoing calcium phosphate maturation while multiple microcalcifications merging into the single macrocalcification indicated ossification. Compact ossifying macrocalcifications with smooth margins frequently had a dense connective tissue capsule. The microenvironment of calcium deposits was often characterised by leaky neovessels. Conclusion . Diversity of calcification morphologies and ossification in atherosclerotic plaques is similar to those observed in calcific aortic valve disease and bioprosthetic heart valve failure. Combined with atherosclerotic plaque disaggregation followed by flow cytometry and singlecell RNA sequencing, our technique may improve our understanding of atherosclerotic calcification and lead to the identification of appropriate therapeutic targets to prevent or retard this process.

X-ray CT Observation of Negative Electrode in Lithium Ion Batteries by Silicone Resin Embedding Method

X-ray CT Observation of Negative Electrode in Lithium Ion Batteries by Silicone Resin Embedding Method

Ultra-low concentration of total organic carbon in ultrapure water using ion-exchange resin embedding silanized magnetic nanoparticles

Regeneration of pure water is an important issue not only for the healthy life but also for the fine control of precise processes in various industries. One important issue in ultra-high purified water is to reduce the amount of total organic carbon (TOC). Herein, we introduce a new approach to reduce the TOC using the surface silanized nanoparticles, in which the magnetic nanoparticles (mNPs) are silanized and then complexed with ion exchange resin (IER) beads. The Fe3O4 mNPs are surface modified by using high concentrated vinyltrimethoxysilane (VTMS) and then adhered on the surface of IER beads. The surface modified mNPs have a thick-shell of polysiloxane layer varying from 5 to 22 nm depending on the amount of VTMS used, which leads the significant increase of specific surface area. The IER beads embedding VTMS-silanized mNPs achieves about 7 μg/L of the TOC level in ultrapure water system, which is two orders less than 228 μg/L of the feeding water and one order less than 96 μg/L from the system using pristine IER beads. This result is mainly attributed to the polysiloxane layer forming broccoli-like surface structure and some part by the vinyl group of VTMS exposed to the amines in the water.